The present invention relates to the field of molecular biology and more particularly to a novel DNA sequence possessing regulated transcriptional promoter activity in two directions, to expression vectors containing this sequence, and to their use for the production of recombinant proteins from either homologous or heterologous sources. The invention also relates to the recombinant cells containing this DNA sequence.
Model bacterial species, such as Escherichia coli, have been used in important molecular biological processes such as the amplification of desirable nucleic acid sequences and the production of heterologous gene products. Unfortunately the ability of bacteria to make functional products from eukaryotic genes is often limited due to alternate prokaryotic codon biases, improper protein folding, and divergent pathways of secondary modifications such as protein glycosylation. For this reason many researchers have turned their attention to yeast species, some of which have been found to be suitable hosts for the expression of proteins of eukaryotic origin.
Yeasts are more easily cultured than cells of many higher eukaryotes and lend themselves to genetic manipulations commonly performed with prokaryotes yet have the capacity for complex post-translational modifications including glycosylation events, proteolytic maturation, and disulphide bond formation (Eckart and Bussineau 1996). Traditionally Saccharomyces cereviseae has been used for this production but in recent years focus has shifted to other yeast species, including the dairy yeast Kluyveromyces lactis, which has become a model system of studies on the molecular physiology of xe2x80x9cnon-conventionalxe2x80x9d yeasts (Breunig et al 2000, Flores et al 2000). One of the difficulties with S. cereviseae as a production system is its preference for fermentation under aerobic conditions. This is known as the Crabtree effect. The Pasteur effect is the opposite: the repression of fermentation in aerobic conditions (Breunig et al 2000).
Crabtree-positive yeasts need to be cultured under sugar limited conditions to prevent the decarboxylation of pyruvate to ethanol. Such conditions typically result in low rates of growth and protein synthesis (Breunig et al 2000). If S. cereviseae is not grown under sugar-limited conditions, each molecule of pyruvate that is reduced to ethanol is not available to the TCA cycle again resulting in reduced biomass yields and ATP production (Schaffrath and Breunig 2000).
Kluyveromyces lactis, like many yeast species, differs from S. cereviseae in that it prefers respiration as opposed to fermentation under aerobic conditions. Under aerobic conditions K. lactis oxidizes pyruvate to CO2 through the TCA cycle and therefore produces more ATP and reducing equivalents per molecule of pyruvate (Flores et al 2000). Besides relief from the Crabtree effect K. lactis possesses a number of other attributes that make it an attractive host for the expression of heterologous proteins. As a naturally occurring dairy yeast, K. lactis is generally regarded as a safe organism and has been considered a food grade organism by the US FDA. It has excellent fermentative characteristics and can be cultured at high cell densities ( greater than 100 g dry cell weight/l) making it amenable to large-scale industrial applications.
Genetic tools have been developed to make use of Kluyveromyces as a host system for the production of recombinant proteins. Several heterologous gene expression systems have been successfully developed for K. lactis making this organism an attractive alternative for heterologous gene expression in industrial applications. Kluyveromyces lactis is capable of using many of the vectors, promoters and marker genes already used in S. cereviseae systems however these have not been optimized for K. lactis. With the exception of natural promoters for the 3-phospoglycerate kinase (U.S. Pat. No. 5,646,012), RP28 ribosomal protein (U.S. Pat. No. 5,627,049), alcohol dehydrogenase (U.S. Pat. No. 5,624,046), transaldolase (U.S. Pat. No. 5,616,474) and pyruvate decarboxylase (U.S. Pat. No. 5,631,143) genes no other endogenous promoters have been developed for expression in K. lactis. The rate at which an introduced gene will be transcribed depends not only on the nature of the gene but also on the promoter associated with the gene. The selection of the appropriate promoter is therefore crucial for efficient heterologous protein expression. Certain applications favor the use of strong constitutive promoters to drive gene expression while other applications require promoters that can be externally regulated to give a phased expression. Regulated expression is particularly useful for the expression of products that may be toxic to the host organism.
Fermentation conditions can be controlled such that the toxin is not produced while host cell biomass is increased. This is commonly achieved by the use of glucose as a major carbon source due to its ability to suppress the expression of genes necessary for the utilization of alternate carbon sources (Dong and Dickson 1997). Regulated expression can be achieved by utilizing promoters derived from one of these alternate pathways. Many of the proteins involved in this type of carbon catabolite repression have been identified in S. cereviseae and most are believed to have functional analogues in K lactis. 
The Saccharomyces ScSNF1p kinase, which has been conserved across many eukaryotic taxa from yeasts, plants and mammals (Dong and Dickson 1997, Carlson 1998), is part of the signal cascade that directs a coordinated response to changing glucose concentrations. One of the targets of ScSNF1p is Mig1p, a DNA-binding transcriptional repressor of many glucose-repressed genes (Lutfiyya and Johnston 1996, Hu et al 1999, Breunig et al 2000). Mig1p binds to conserved GC-elements present in the promoters of glucose-repressed genes and disruption of Mig1p gene function results in dramatic de-repression of expression (Wang et al 1997, Zaragosa et al 2000). Kluyveromyces lactis possess a Mig1p homologue (KlMig1p) responsible for the repression of the glucose-repressed lactose-galactose regulon (Dong and Dickson 1997, Hu et al 2000). De-repression is not complete in the absence of KlMig1p suggesting the presence of an additional KlMig1p independent pathway and regulation by other repressors (Dong and Dickson 1997). Glucose repressed genes in alternative sugar metabolism also involve transcriptional activator proteins. In Saccharomyces, Gal4p is a transcriptional activator of many glucose-repressed genes and is turn repressed by Gal80p. This secondary repression is relieved by raised intracellular concentrations of non-glucose sugars (Hu et al 2000). The K. lactis homologue KlGal4p (also called Lac9p) escapes inhibition by KlGal80p and is thought to activate its own expression, thereby increasing the concentration of the activator. The concentration of KlGal4p (Lac9p) limits expression of many glucose repressed regulons. In addition to KlMig1p and KlGal4p(Lac9p) there are other transcription factors, many of which are yet to be identified, that add specificity to each of the regulated alternate pathways.
The selection of an appropriate promoter is crucial for efficient heterologous protein expression. Although relatively rare, bi-directional promoters are generally believed to regulate related gene products that are produced in stoichiometric quantities (Levine et al 1992, Menne et al 1994). An artificial construct utilizing the bi-directional expression of the reporter genes gusA and lacZ, driven by the promoter of a-aminoadipyl-cysteinyl-valine [ACV] synthetase/isopenicillin N-synthetase from Acremonium chrysogenum, has shown that the pcbC promoter (isopenicillin N-synthetases) was at least 5 times stronger than pcbAB (ACV synthetase) promoter (Menne et al 1994).
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Carlson M. 1998 Regulation of glucose utilization in yeast. Current Opinions in Genetics and Development 8:560-564.
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Lutfiyya L L, Johnston. 1996. Two zinc-finger-containing repressors are responsible for glucose repression of SUC2 expression. Molecular and Cellular Biology 16:4790-4797.
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In accordance with the instant invention, what has been identified, cloned and sequenced is a region of the K. lactis genome having regulated transcriptional promoter activity [SEQ ID NO: 1]. This sequence was obtained by screening a total genomic library of K. lactis with a probe derived by reverse genetics of a highly expressed gene product followed by sequence analysis of the entire gene locus. More specifically the region shown in [SEQ ID NO: 1] corresponds to the promoter of the K. lactis maltase gene and may be used for the efficient production of recombinant proteins in yeasts of the Kluyveromyces genus and may also be used in other host organisms.
Moreover analysis of a larger region of the Kluyveromyces genome, immediately flanking [SEQ ID NO: 1], has identified two reading frames in opposite directions; one corresponding to the maltase gene and the other corresponding to the maltose permease gene. This unexpected discovery demonstrates that the complementary strand of the region presented in [SEQ ID No: 1] also possesses promoter activity acting in the opposite direction [SEQ ID NO: 2].
One aspect of the present invention involves the DNA sequence comprising the sequence presented in [SEQ ID NO: 1], or a sequence of its complementary strand presented in [SEQ ID NO: 2], or derivatives thereof which possess promoter activity.
Derivative is understood to mean any sequence obtained from the sequences given in [SEQ ID NO: 1] and [SEQ ID NO: 2], by structural modifications (mutations, additions, deletions, substitutions) which conserve or modify promoter activity in either direction. In particular, the mutations may involve one or more nucleotides and the additions, deletions, or substitutions may involve regulatory elements or binding sites for positive activators.
Another aspect of the present invention relates to a recombinant DNA comprising a DNA sequence as defined above. This recombinant DNA may contain the promoter sequence, or a derivative thereof, in which one or several restriction sites have been advantageously inserted, or to which a multiple cloning site has been added, to facilitate the insertion of one or more structural genes.
More preferably, the recombinant DNA contains signals permitting the sub-cellular targeting, surface localization, or secretion of the expression products of said structural genes.
In a specific embodiment of the invention, the recombinant DNA is part of an expression plasmid that may be autonomously replicating or integrative in nature.
In particular, autonomously replicating vectors may be ideally obtained by using autonomously replicating sequences (ARS) in the host selected. In K. lactis replication origins from known plasmids (such as pKDI) or a novel ARS may be advantageously involved.
Integrative vectors may be obtained by using homologous sequences corresponding to certain regions of the host genome which permits targeting of the vector to a specific locus by integrative recombination.
Another aspect of the present invention relates to recombinant cells that contain at least one of the DNA sequence as defined above.
For such purpose, Cells are chosen preferably from yeasts of the Kluyveromyces genus however it is understood that the invention is applicable to all recombinant cells, including but not limited to all yeasts and filamentous fungi, in which the promoter regions of the invention are active.
These cells may be obtained by any one of well known methods for enabling a foreign DNA to be introduced into a cell. For example, this may be accomplished by cell protoplast transformation, electroporation, ballistic gene delivery or any other technique known to a person skilled in the art.
Another aspect of the invention relates to the use of the sequence as defined above for the expression of recombinant genes. In particular, it is possible to use these sequences to simultaneously drive the expression of several genes.
Advantageously, the invention permits the use of the bi-directional promoter for the simultaneous expression of recombinant genes in opposite directions. These genes may be tandemly linked as fusion proteins in a single direction and two sets of genes may be expressed in opposite directions.
Advantageously, one of the genes may encode an easily assayed reporter while the other gene may encode a product for which no assay exists. Expression levels of the easily assayed reporter can be used to assess the relative expression of the partnered gene.
Advantageously, one of the genes may encode a positive activator for the enhanced transcription from the opposite promoter such that cellular levels of positive activator for the optimal expression from the opposite promoter are not limiting.
Advantageously, the sequences of the invention may be used for the expression of genes encoding proteins of interest in the industrial enzyme, pharmaceutical, or foodstuffs sectors. By way of example, there may be enzymes such as polygalacturonase, xylanase, phytase, superoxide dismutase, catalase, amylase, cellulases, chitinase, and chymosin, N-glycan processing enzymes such as mannosidase, mannosyl transferase, N-acetyl glucososaminyl transferase, galactosyl transferase, and sialyl transferase, hydrophobic polypeptides such as cerato-ulmin, hydrophobin a and b, lymphokines such as interleukins, interferons, and colony stimulating factors, growth factors such as growth hormones, erythropoietin, FGF, EGF, and TGF, antigenic polypeptides for the production of vaccines or alternately polypeptide fusions of portions of antigenic polypeptides with stabilizing molecules.